The single objective of this grant application is to establish methodology in a mouse model for projected, subsequent clinical pharmacology studies for specifically quantifying 4-hydroxyifosfamide (HOIfos), the activated, latent-alkylating, cytotoxic metabolite of the clinically-used antitumor drug ifosfamide (Ifos), in plasma of patients treated with -Ifos by comparing a reported method, which does not require radiolabeled drug but quantifies HOIfos non-specifically by trapping acrolein liberated by this metabolite as it decomposes to aldoifosfamide (aldoIfos) and subsequently to acrolein and isophosphoramide mustard (IPM), the ultimate alkylating and cytotoxic metabolite of Ifos, with a HOIfos-specific thin layer chromatography (TLC) method, which requires use of radiolabeled drug. Because humans and experimental animals extensively metabolize Ifos to its two monodechloroethylated derivatives (dechloroethylIfos and dechloroethylcyclophosphamide), it is possible and likely that the latter metabolites are further metabolized to their 4-hydroxy derivatives, which would also decompose to yield acrolein and monodechloroethylated-IPM, yielding elevated but erroneous levels of HOIfos via use of the acrolein- trapping methodology. The chief, potential contribution that the proposed investigations in mice could make would be to demonstrate that clinical pharmacology of ifosfamide will not require use of radiolabeled drug for accurate AUC data for HOIfos.